DE1795282A1 - Process for the production of S-adenosyl homocysteine - Google Patents

Description

Patent attorneys Dipl.-Ing. F. Weickmann, Dr. Ing. A, Weickmann

Dipping. H. Weickmann, Dipl.-Phys. Dr. K. Fincke Dipl.-Ing. F. A. Weickmann, Dipl.-Chem. B. Huber

8 MUNICH 27, MOHLSTRASSE 22. CALL NUMBER 4IJ921 / 22

BOEHRIIiGER HAOHEIH GMBH Mannheim

Process for the production of S-adenosyl homocysteine

The invention relates to a chemical process for the production of S-adenosyl homocysteine by demethylation of S-adenosyl methionine.

• S-adenosyl-L-homocysteine (SAH) is an important biological one Intermediate product that occurs in all transmethylation reactions in which S-adenosyl methionine (SAM) is involved in nature is. This methyl group transfer is fundamental for the entire organism (e.g. hucleic acid and amino acid metabolism) Significance and justifies the considerable interest in the connections SAM and SAH involved in these conversions. For example SAH is a reaction product in the biosynthesis of methionine, gholin, metadrenaline, adrenaline, N-methyltetrahydrofol-

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acid, anserine, L-methylhistamine, N-methy! nicotinamide, creatine, Spermidine, spermine and various alkaloids.

SAH has so far been obtained either by chemical synthesis (W.Sakami, Biochem. Prep. Vol. 8, 8); by isolation from yeast (Shapiro, Anal. Biochem. t ^, 323 (1966)); by enzymatic demethylation of SAM (Shapiro et al., J. biol. Chem. 2 £ 2, 1551 (1964), Haba, Cantoni, J. biol. Chem. 234 , 603 (1959) »Duerre, Arch. Biochem. Biophys . Vol. <} 6, 570 (1962)) or by enzymatic synthesis from adenosine and homocysteine with liver enzyme (Duerre, Schlenk, Arch. Biochem. Biophys. Vol., 6, 575 (1962), but all of these methods are presented very labor-intensive and not very satisfactory in terms of yield. The enzymatic intmethylation processes suffer from the inaccessibility and instability of the enzymes involved. Since there was no technically usable production process, SAH was not commercially available either.

The invention was therefore based on the object of a simple, technical to create a viable method of making SAH from the readily available SAM.

The solution to this problem was countered by the difficulty that because of the great sensitivity of SAM to chemical agents, those in preparative organic chemistry for sulfonium compounds otherwise customary fragmentation reactions for converting SAM into SAH cannot be used (see Houben-Weyl, Vol. 9, 175).

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Eb is known to have S-adensyl methionine (SAM; Formula 1), depending on the reaction conditions, according to A, B, G, D and E is degraded;

-CH ₀ -CH - COOH ² I.

OH OH

The bond A is preferably cleaved in an acidic medium, that of B, C and E "preferably in an alkaline medium, while the realization of the reaction D was only enzymatically but not chemically feasible up to now. So writes S. Sohlenk, a pioneer in this field, in "Q? He Chemistry of Biological Sulfonium Compounds" (Advances in chemical-organic natural substances, Vol. 23t 1965, p. 75):

"the bond between the sulfur atom and the methyl group (D in formula 1) shows particular resistance; so far we could no hydrolysis conditions are found that differ from S-adenosyl methionine lead to S-adenosyl homocysteine ".

The only one known so far (Krollpfeifer, Chem. Reports, year 86, No. 9, p. 1049) and using the specific example of the cleavage of diarylalkylsulfonium salts to diaryl sulfides described

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method for converting sulfonium salt into sulfide fails in the Fall of SAM in SAH completely. When exposed to alkali chlorides in aqueous medium on SAM at the boiling point, as described in this process, only methyl adenosyl sulfide (MAS) is used received (according to route A).

Surprisingly, it has now been found that under certain conditions a chemical conversion from SAM to SAH, d. H. Cleavage of bond D in Formula 1 without substantial cleavage of the bonds A, B, C or E, possibly isb.

The process according to the invention for the production of S-adenosyl-homocysteine consists in that in a polar organic solvent or solvent mixture an S-adenosyl-methionine salt soluble therein at temperatures between about 10 and 10O ⁰ G with an alkali or alkaline earth metal halide or pseudohalide is implemented.

A particularly suitable polar organic solvent is a solvent containing building materials, such as one used in the Reaction temperature liquid alcohol, a lower fatty acid or a lower ketone. Preference is given to alcohols and ketones and fatty acids having 1 to 4 carbon atoms are used. Methanol is particularly preferred. The solvent used must be practical be anhydrous. Preferably, any water present is formed with the formation of a suitable azeotrope after the dissolution of the SAM salt distilled off. For example, im

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If methanol is used as the solvent, the azeotrope water / methanol / benzene.

The type of SAM salt used depends on the solvent used since the different SAM salts have different solubility in different organic solvents. examples for suitable SAM salts are formate, acetate, perchlorate, chloride, Bromide and iodide. Because of their good solubility, iodide and chloride are preferred.

The SAM-SaIz used does not have to be in pure form. For example Commercially available raw SAM salt with a SAM content of about 20 to 25 # is suitable for the process of the invention. Since commercial SAM salt is often in the form of sulfate, which does not have the desired solubility, an anion exchange is advantageously carried out by reaction with a suitable barium salt, e.g., barium iodide. The barium sulfate formed during the reaction leaves then easily move away.

Alkali or alkaline earth metal halides suitable as demethylating agents or pseudohalides can be, for example, the salts of lithium, sodium, potassium, ammonium, magnesium, calcium, strontium and barium with halogen, dan ions such as chloride, bromide, iodide or call pseudohalide anions such as rhodanide. Preferred are the Iodides, as they are, on the one hand, particularly highly organosoluble and, on the other hand, particularly suitable for demethylation.

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The halide is related to the SAM-SaIz used for the reaction at least in equimolar amounts, but preferably in excess. Suitable SAM: halide molar ratios are between about 1 and 10, the halide is preferably used in a molar excess. When using barium iodide For example, it is possible to convert SAM sulfate into SAM iodide and react with the alkaline earth metal halide in one Carry out one-step reaction.

The reaction of the invention proceeds, as mentioned, at temperatures between 10 and 100 ⁰ C at a reasonable rate, when using an alkali metal or Erdalkalijodides is preferably carried out at a temperature between 45 and 55 ⁰ C, because at this temperature the methyl iodide formed distilled off and so that the heating equilibrium is constantly shifted in the direction of SAH. At a temperature of 50 ^° C., the reaction time is about 24 hours until conversion is complete, ie until SAH can no longer be detected. This results in some MAS and homocysteine as a by-product (according to route A in formula 1). Fewer by-products are formed at lower temperatures, but the reaction proceeds very slowly. At a higher temperature, the rate of the reaction, but also the formation of by-products, increases considerably.

The recovery of pure SAH from the process according to the invention resulting solution is advantageous in a simple manner.

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carried out by adsorption on activated carbon and desorption with alkaline aqueous-alcoholic solution. Is particularly suitable here the use of a coal column. Crystallized from the eluate Looked out at neutral pH and left standing in the cold.

The process according to the invention can be carried out in a simple manner without any particular expenditure on equipment. The yield is good and can be 80 fa or more, based on the SAH used.

The following examples further illustrate the invention.

example 1

in/

200 g raw SAM. HSO. (SAM content approx. $ 20 to 25) are 500 ml Dissolved distilled water and mixed with a concentrated aqueous solution of 200 g of bariungodide. The failed precipitate the barium sulfate is centrifuged off, washed once with distilled water and centrifuged again. The United Supernatants are concentrated in vacuo to an oily consistency and, with thorough stirring, in a mixture of 2.5 l of methanol and Poured 500 ml of benzene. Without removing the precipitate which has precipitated out, the suspension is concentrated to approx. 2 liters in vacuo and the concentrate is diluted to 3 liters with absolute methanol. The yellowish solution is in the water bath for 24 hours kept at 50 °, cooled to room temperature and chromatographed on a carbon column (length 1.20 m, diameter 4 cm) and with 4 liters of distilled water nacji ^ ev / ash.

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The drawn SAH is removed from the coal column with ethanol / water / concentrated ammonia (50: 50: 1) eluted. The absorbent in UV light at 260 πιμ Fractions are collected and concentrated in vacuo to about 200-300 ml. The pH of the concentrate will be adjusted to 7 with acetic acid. The SAH is then allowed to crystallize out at 0 °. After 2 days the crystalline SAH is sucked off, washed with a little ice-cold water and made from hot distilled water. Recrystallized water.

Yield: 30 to 35 g of SAH. 2 H ₂ O (60 to 71 fi of theory, based on SAIi used).

Example 2

200 g raw SAM. HSO. λ-rerden dissolved in 500 ml of formic acid and mixed with a solution of 150 g of NaI in 2.5 l of methanol. Without the precipitate to be removed, the suspension is incubated for 24 hours at 50 ⁰ O and worked up further as described in Example. 1

Yield: 30 to 35 g as dihydrate.

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Claims (7)

- Q _ claims 1. A process for the preparation of S-adenosyl homocysteine by demethylation of S-adenosyl methionine, characterized in that a soluble therein S-adenosyl methionine salt at temperatures between about 10 and 100 ⁰ O with a polar organic solvent or solvent mixture Alkali or alkaline earth halide or pseudohalide is reacted. 2. The method according to claim 1, characterized in that as Solvent an alcohol, ketone or a fatty acid with 1 up to 4 carbon atoms is used. 3. The method according to claim 2, characterized in that methanol is used. 4. The method according to any one of claims 1 to 3 »characterized in that that the chloride or iodide is used as the soluble S-adenosyl methionine salt. 5. The method according to any one of claims 1 to 4, characterized in that that an alkali or alkaline earth iodide is used. 6. The method according to any one of claims 1 to 5, characterized in that the reaction at a temperature between 45 and 55 ⁰ O is carried out. 209816/1722 " ¹⁰ " 7. The method according to any one of the preceding claims, characterized in that a ratio of S-adenoeyl methionine salt to alkali or alkaline earth halide or pseudohalide between 1: 1 and 1:10, preferably 1: 2, is used. 209816/1722